Genetic recombination has enabled mass-production of a protein which has heretofore been extremely difficult to utilize as existing only in a minor amount in living bodies and therefore difficult to isolate, and a polypeptide having any desired amino acid sequence, using bacteria or animal cells as a host. Various bacteria are used as a host for protein production through genetic recombination, and those that are most widely used are Escherichia coli. However, in the recombinant protein production system using Escherichia coli as a host, since the produced protein is accumulated in the cells, the cell volume is the uppermost limit of the produced protein and therefore mass production of protein is difficult. Further, there are other problems in that the Escherichia coli cells must be disrupted for the purpose of collecting the protein accumulated in the cells, and the operation for isolating and recovering the intended protein from the cell-derived component, nucleic acid, other proteins than the intended one, and the cell wall-derived endotoxin that are contained in the disrupted Escherichia coli cells is troublesome.
To solve the problems with Escherichia coli systems, a recombinant protein production system has been developed in which Bacillus subtilis, typical bacteria having the ability to secrete and produce protein, is used as a host. In the production system where Bacillus subtilis is used as a host, the produced recombinant protein is secreted and produced in the medium. Therefore, the Bacillus subtilis system has some excellent properties that the Escherichia coli system does not have, in that the former does not require cell disruption for protein recovery and the former gives little endotoxin.
On the other hand, however, Bacillus subtilis has the property that it extracellularly secretes a large amount of protease, and therefore, there exists a serious problem with it in that the secreted and produced recombinant protein is degraded by the protease and the yield of the recombinant protein is extremely low. Accordingly, various efforts have heretofore been made for reducing the production of the protease in Bacillus subtilis. Nevertheless, however, there have been to date known few examples of using a recombinant protein production system with Bacillus subtilis as a host for industrial production of heterogeneous proteins.
On the other hand, to solve the problem with the Bacillus subtilis system, a new recombinant protein production system has been developed in which are used bacteria capable of extracellularly secreting and producing a recombinant protein but not extracellularly secreting a protease, as a host. As a result, a recombinant protein production system has been successfully developed in which is used Bacillus brevis such as Bacillus brevis 47 (Patent Reference 1) that exhibits better protein secretion productivity than Bacillus subtilis, as a host.
At present, as a result of systematic analysis thereof based on the base sequence of 16SrRNA gene, Bacillus brevis is re-classified as bacteria of the genus Brevibacillus that includes Brevibacillus choshinensis, Brevibacillus brevis (Non-Patent Reference 1).
However, it has been found that Bacillus brevis 47 has a weak extracellular proteolytic activity. It is said that recombinant protein production using Brevibacillus brevis as a host takes a relatively long period of time (generally 3 days or so). Accordingly, even though the extracellular proteolytic activity of Bacillus brevis cells is weak, there may be a case where the produced recombinant protein may be degraded by the extracellular proteolytic activity and the yield of the recombinant protein may reduce. To solve this problem, there have heretofore been made various efforts for further reducing the extracellular proteolytic activity of Bacillus brevis. 
For example, Bacillus brevis strains have been screened in a broad range, and, as those having an extremely low extracellular proteolytic activity and having an excellent protein secretion productivity, there have been isolated Brevibacillus choshinensis HPD31 (FERM BP-1087) (the same strain as that of FERM BP-6863: Bacillus brevis H102) (Patent Reference 2), and its mutant strain, Brevibacillus choshinensis HPD31-S5 (FERM BP-6623—this strain has been deposited as a designation of Bacillus brevis HPD31-S5) These strains have been utilized as a host in production of various recombinant proteins such as human epidermal growth factor (hEGF). For example, production of hEGF with Brevibacillus choshinensis HPD31 is shown in Non-Patent Reference 2 (in this, Brevibacillus choshinensis HPD31 is described as Bacillus brevis HPD31), etc.
However, even these Brevibacillus choshinensis HPD31 and HPD31-S5 showed extracellular proteolytic activity when analyzed in a high-sensitivity proteolytic activity detection method. For example, analysis of the culture supernatant of Brevibacillus choshinensis HPD-31 through gelatin-PAGE (zymograph) for detection of proteolytic activity thereof gave a gelatin decomposition band as in FIG. 2.
On the other hand, Bacillus brevis 31-OK not substantially showing a protease activity (proteolytic activity), referred to as a Bacillus brevis mutant strain, is disclosed (Patent Reference 3). However, even the Bacillus brevis 31-OK gave a band showing its proteolytic activity in its test for evaluation of the proteolytic activity thereof according to the gelatin PAGE method, and it did not completely lose the extracellular proteolytic activity. Moreover, the gene level analysis of the extracellular proteolytic activity of the strain has not been attained at all. Specifically, the protease was not isolated, and, needless-to-say, not only the sequencing of the gene thereof but also even the cloning thereof was not carried out.
Accordingly, even in the case of using strains of which the extracellular proteolytic activity has been said to be reduced, there may be a possibility that the secreted and produced protein may be degraded by the extracellular proteolytic activity that the strain still have, and therefore the yield of the protein may reduce.
These strains of Brevibacillus choshinensis (or Bacillus brevis) were obtained through screening or mutation by treatment with a mutagen, or other means, but were not through identification of the protease gene on the genome further followed by inactivation of the gene. Accordingly, to date no one knows Brevibacillus choshinensis of which the proteolytic activity has been reduced through identification of the protease gene on the genome further followed by inactivation of the gene.
The previously-described reduction in the proteolytic activity of Brevibacillus choshinensis is entirely for the reduction in the extracellular proteolytic activity thereof, and no attention has been paid to the reduction in the intracellular proteolytic activity of the strains. However, in recombinant protein production using Brevibacillus choshinensis as a host, there is a case where secretion production is impossible but intracellular accumulation production is possible depending on the type of the intended protein. In such a case, the produced protein may be decomposed by the action of the intracellular protease and, as a result, a recombinant protein is not almost obtained.
It is known that cells of Brevibacillus choshinensis may partly undergo bacetriolysis during culture, and, as a result, the intracellular protein that contains the intracellular protease may dissolve out into the medium. Accordingly, there is a possibility that even the recombinant protein extracellularly secreted and produced may be degraded by the dissolved intracellular protease.
Therefore, reducing the intracellular proteolytic activity is also another problem that is to be solved for the purpose of further increasing the usefulness of Brevibacillus choshinensis as a host in genetic recombination.
Heretofore, there has been known no example of reducing the intracellular proteolytic activity of Brevibacillus choshinensis. 
In addition, gene-level analysis of Brevibacillus choshinensis for the intracellular proteolytic activity thereof has not been carried out at all. Specifically, there has been known no example of isolating the intracellular protease of Brevibacillus choshinensis. In addition, there have also been known no example of cloning the intracellular protease gene and no example of determining the sequence thereof.
Moreover, for the purpose further broadening the industrial utilization of Brevibacillus choshinensis as a host in genetic recombination, there is still another problem to be solved, apart from the reduction in the proteolytic activity of the strain.
Brevibacillus choshinensis may form spores, like bacteria of the genus Bacillus such as Bacillus subtilis. As compared with living cells (non-spore cells), spores have high heat resistance and therefore require extremely severe conditions for sterilization. Accordingly, in production of recombinant protein pharmaceuticals that require a guarantee of complete sterilization and removal of cells including spores in production lines after completion of culture, an extremely difficult technique is necessary for producing them by the use of Brevibacillus choshinensis as a host.
Accordingly, for the purpose of making it possible to utilize Brevibacillus choshinensis as a recombination host in broad-range industrial applications such as production of recombinant protein pharmaceuticals, desired are Brevibacillus choshinensis strains that do not form spores and can be completely killed even under mild sterilization conditions (from 60° C. to 100° C.)
The condition necessary for sterilizing the spores of Brevibacillus choshinensis is much milder than the condition necessary for sterilizing the spores of Bacillus subtilis. For example, it is said that the D value of the spores of Bacillus subtilis at 100° C. (the time taken for reducing the number of all cells including living cells (non-spore cells) and spores to 1/10 in a different temperature range) will be 11 minutes or so, though varying depending on the type of the strain. On the other hand, the D value of the spores of Brevibacillus choshinensis is at most 1 minute. However, the sterilization of the spores of Brevibacillus choshinensis requires extremely severer conditions as compared with the sterilization of the living cells (non-spore cells) thereof. For example, as demonstrated in Examples given hereinunder, the D value of the living cells (no-spore cells) of Brevibacillus choshinensis at 80° C. is shorter than 1 minute, but the D value of the spores thereof is 67 minutes or so.
Bacillus subtilis SMS275 (Patent Reference 4: JP-A 4-287686 (1992)) is disclosed as anon-spore strain of Bacillus subtilis. However, there has heretofore been unknown any strain of Brevibacillus choshinensis not having the ability to form spores, or that is, capable of being completely killed under mild sterilization conditions.
Patent Reference 1:
JP-A 60-58074 (1985)
Non-Patent Reference 1:
Int. J. Syst. Bacteriol., 46, 939-946 (1996)
Patent Reference 2:
JP-A 63-56277 (1988)
Non-Patent Reference 2:
Ann. NY Acad. Sci., 782, 115-122 (1996)
Patent Reference 3:
JP-A 6-296485 (1994)
Patent Reference 4:
JP-A 4-287686 (1992)